This invention relates to a method for using non-growing, metabolically active methylotrophic yeasts to convert natural source primary alcohols to natural flavour aldehydes.
There is a consumer preference for natural products; such that food products labelled as containing "all natural flavours" have a market advantage over similar products containing artificial flavours. This preference has lead to a demand for natural flavours, but supply is limited. Accordingly, natural flavours command a much higher price than similar synthetic flavours.
Several factors tend to force the product price higher. Nearly all natural flavours are obtained as extracts from botanical sources, but plant materials often contain low concentrations of the desired flavour compound making extraction expensive. Also, the supply of raw materials is subject to seasonal and climatic variation, while in some cases socio-political instabilities in a producing region may threaten supply.
Many yeasts, molds and bacteria produce metabolites with flavour and fragrance attributes. In the United States, the Code of Federal Regulations states that products produced or modified by living cells or by their components, including enzymes, may be designated as "natural". Many flavours may therefore be produced by biotechnological means and marketed as natural in the U.S. The development of microbial fermentation technologies should increase the availability of many natural flavours, and thereby assure uniform quality and constant supply.
When methylotrophic yeasts of the genera Pichia, Torulopsis, Candida and Hansenula are grown using methanol as the sole source of carbon, subcellular vesicles known as peroxisomes are formed. These microbodies are the location of the enzyme alcohol oxidase, the first enzyme in the dissimilatory pathway that enables the organism to use methanol as the sole carbon source for growth. The enzymes of this pathway (shown below) have been purified and studied in detail by a number of workers. ##STR1##
Alcohol oxidase is under a repression/depression type of metabolic control system (H. Sahm, Adv. Biochem. Eng. 6, 77, 1973). Growth of the organism on soluble carbohydrate sources such as glucose prevents the formation of peroxisomes. During adaptation of the organism from growth on glucose to methanol, alcohol oxidase activity increases before growth is able to resume (H. Sahm and F. Wagner, Eur. J. Biochem 36, 250, 1973).
Alcohol oxidase is of particular interest as a biotechnological tool because it is relatively nonspecific and because it is stable over a useful range of reaction conditions. A number of potential uses have been suggested for alcohol oxidase. These include use of the enzyme in a quantitative assay for alcohol, as an oxygen scavenger, for sterilization (through the release of formaldehyde) of heat- or radiation-sensitive materials, for the production of flavouring compounds (Alcohol oxidase product brochure, Provesta Corp., Bartlesville, Okla.), and as part of an ethanol recovery system (M. Kierstan, Biotechnol. Bioeng. 24, 2275, 1982). All of these applications have made use of cell-free extracts of varying purity as a source of alcohol oxidase. Other workers have succeeded in using both free and immobilized whole cells of Hansenula polymorpha for the production of formaldehyde from methanol (J. Baratti et al, Biotechnol. Bioeng, 20, 333, 1978; R. Couderc and J. Barati, Biotechnol. Bioeng. 22, 1155, 1980).
We have found whole cells of Pichia pastoris can be used with advantage in place of purified alcohol oxidase for the production of flavour aldehydes. Whole cells have a number of advantages over cell-free enzymes. Intracellular enzymes are protected from changes in conditions such as pH and ionic strength which may occur in the reaction vessel. As well, essential cofactors and coenzymes (such as FAD and catalase in the case of alcohol oxidase) are "co-immobilized" with the enzyme of interest, facilitating multistep reaction mechanisms. In this invention, a model system for aldehyde production was developed, based on the conditions for optimum conversion of ethanol to acetaldehyde.
In the production of flavour aldehydes a major limitation is the problem of end-product inhibition. It is known (U.S. Pat. No. 4,481,292) that this problem can be partially alleviated by chelating the aldehyde with alkaline Tris buffer. However, we have found that this complexing process is limited by the release of H.sup.+ and a subsequent drop in pH. Lower pH results in decreased Tris-aldehyde binding and a return to toxic aldehyde effects. It has been found that the problems with using the Tris buffer can be avoided by using a dual buffering system. The function of the Tris remains to bind the aldehyde while the additional buffering agent maintains the pH close to 8. Alternately other amine buffers can be used which chelate aldehydes but maintain the pH near optimum levels.
U.S. Pat. No. 4,617,274 discloses a method of culturing yeast strains to produce a high cell density and the subsequent use of these cells for carrying out enzyme conversions. For example, methylotrophic yeasts can be used for the conversion of C.sub.3 -C.sub.6 secondary alcohols to their corresponding methyl ketones using secondary alcohol dehydrogenase (SADH) (U.S. Pat. Nos. 4,241,184 and 4,266,034). Primary alcohols are, however, not oxidized by SADH.
U.S. Pat. No. 4,619,898 discloses a new alcohol oxidase isolated from Pichia-type organisms, which can be used in the production of aldehydes and hydrogen peroxide. The advantage of using a whole cell system over the purified enzyme has already been discussed.